The general aim of this project is to elucidate, at the molecular level, how heparin accelerates the inhibition of thrombin and factor Xa by antithrombin III. Rapid reaction kinetics, using stopped-flow and rapid quenching techniques, will be used extensively. We will attempt to quantitatively evaluate whether rate enhancement is due to approximation of the proteins on the heparin, a conformational change of antithrombin making it more reactive, or both. Intermediates in the protease neutralization reaction will be determined, as well as the rate limiting step in the catalytic turnover of heparin under various conditions. Kinetic properties of heparin action on factor Xa inactivation will be compared with those for thrombin neutralization, and the differential effects of heparin size evaluated. This project should result in increased understanding of how heparin increases the affinity between antithrombin and coagulation proteases, resulting in rate acceleration of inactivation.